Project Summary: In the treatment of cancer, fertility-sacrificing chemotherapy and radiation are commonly used treatment regimens. This is particularly alarming for prepubertal males for whom fertility preservation is not offered. Sperm banking is the only current procedure for male fertility preservation; however, this technique does not cover prepubertal males, who have no sperm to collect. This clinical gap must be addressed by basic science discoveries into in vitro spermatogenesis and testis modeling. Proper compartmentalization of testis cell types into seminiferous tubules and interstitial spaces is necessary for spermatogenesis. In addition, tubule formation coincides with endothelial cell migration into the developing testis, without which there is no tubulogenesis. In vivo techniques have successfully restored spermatogenesis through spermatogonial stem cell transplantation, and testis cell subcutaneous transplantation, which recapitulates de novo tubulogenesis. In vitro techniques using collagen hydrogels as an artificial extracellular matrix (ECM), and air-liquid interface culture have also created de novo testis tubules from aggregated testis cells. However, challenges remain with these methods; namely a low yield of tubules containing meiotic germ cells, a lack of post-meiosis germ cell differentiation, and an understudied endocrine capacity of interstitial Leydig cells. Moreover, to my knowledge, there have been no published studies directly comparing different ECMs for testis modeling, nor incorporating a functional vasculature. The central hypothesis of this proposal is that ECM-based cues and vascular signals are necessary for optimal in vitro testis compartmentalization, germ cell expansion and differentiation, and interstitial cell endocrine function. In my preliminary studies, I have characterized and decellularized mouse and human testis ECM, which is the predicate for my work proposed in aim 1. Additionally, I have preliminary studies creating biomimetic environments that support endothelial cell proliferation and angiogenesis, which is the predicate for my work in aim 2. In aim 1, I propose to directly compare the efficacy for in vitro testis culture of different hydrogels (more versus less similar to native testis ECM). Success in modeling will be determined via the collection of four data types: (1) germ cell proliferation and differentiation, (2) tubule formation, (3) endocrine function and responsiveness to hormonal stimulation, and (4) germ cell fertilization leading to live birth of pups. In aim 2, I will elucidate the role of vasculature signaling in testis modeling through the co-culture of testis cells with endothelial cells in novel microfluidic systems that reliably produce angiogenesis. The combination of these hydrogel and microfluidic culture techniques will test my overall hypothesis, while additionally creating an in vitro model for testis development and spermatogenesis. Taken together, these basic science discoveries will inform future clinical practice and provide foundational knowledge for the development of assisted reproductive technologies for prepubertal male cancer patients.